Kaempferol, the melanogenic component of Sanguisorba officinalis, enhances dendricity and melanosome maturation/transport in melanocytes.

Kaempferol, a representative flavonoid constituent of Sanguisorba officinalis, promotes melanogenesis, but the underlying mechanisms remain unknown. Here, we evaluated the effects of kaempferol on melanocytes morphology and behavior and determined the mechanisms regulating kaempferol-induced pigmentation. We observed that kaempferol increased melanin contents and dendritic length and stimulated melanocyte migration both in vitro and vivo. It significantly enhanced the expression of microphthalmia-associated transcription factor (MITF) and downstream enzymes of melanin biosynthesis-tyrosinase (TYR), tyrosinase-related protein (TRP-1), and dopachrome tautomerase (DCT). It also induced melanosome maturation (increased stage III and IV melanosomes) and melanin transfer to dendritic tips; this was evidenced as follows: kaempferol-treated melanocytes exhibited the perimembranous accumulation of HMB45-positive melanosomes and increased the expression of Rab27A, RhoA, and Cdc42, which improved melanosome transport to perimembranous actin filaments. These results jointly indicated that kaempferol promotes melanogenesis and melanocyte growth. Additionally, kaempferol stimulated the phosphorylation of P38/ERK MAPK and downregulated p-PI3K, p-AKT, and p-P70s6K expression. Pre-incubation with P38 (SB203580) and ERK (PD98059) signaling inhibitors reversed the melanogenic and dendritic effects and MITF expression. PI3K/AKT inhibitor augmented kaempferol-induced melanin content and dendrite length. In summary, kaempferol regulated melanocytes' dendritic growth and melanosome quantity, maturation, and transport via P38/ERK MAPK and PI3K/AKT signaling pathways.

Catabolism of lysosome-related organelles in color-changing spiders supports intracellular turnover of pigments.

Pigment organelles of vertebrates belong to the lysosome-related organelle (LRO) family, of which melanin-producing melanosomes are the prototypes. While their anabolism has been extensively unraveled through the study of melanosomes in skin melanocytes, their catabolism remains poorly known. Here, we tap into the unique ability of crab spiders to reversibly change body coloration to examine the catabolism of their pigment organelles. By combining ultrastructural and metal analyses on high-pressure frozen integuments, we first assess whether pigment organelles of crab spiders belong to the LRO family and second, how their catabolism is intracellularly processed. Using scanning transmission electron microscopy, electron tomography, and nanoscale Synchrotron-based scanning X-ray fluorescence, we show that pigment organelles possess ultrastructural and chemical hallmarks of LROs, including intraluminal vesicles and metal deposits, similar to melanosomes. Monitoring ultrastructural changes during bleaching suggests that the catabolism of pigment organelles involves the degradation and removal of their intraluminal content, possibly through lysosomal mechanisms. In contrast to skin melanosomes, anabolism and catabolism of pigments proceed within the same cell without requiring either cell death or secretion/phagocytosis. Our work hence provides support for the hypothesis that the endolysosomal system is fully functionalized for within-cell turnover of pigments, leading to functional maintenance under adverse conditions and phenotypic plasticity. First formulated for eye melanosomes in the context of human vision, the hypothesis of intracellular turnover of pigments gets unprecedented strong support from pigment organelles of spiders.

A role for Dynlt3 in melanosome movement, distribution, acidity and transfer.

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on ß-catenin activity, revealing a function of the Wnt/ß-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer.

The role of autophagy in skin pigmentation.

Hyperpigmentation and hypopigmentation are two manifestations of skin pigmentation diseases. Recent studies have shown that autophagy is involved in the development of skin pigmentation diseases. The melanosome is a lysosome-related organelle characterized by the production of melanin. The autophagosome-lysosome degradation pathway exhibits a characteristic cell renewal function. The functions of melanosomes and autophagosomes intersect and the vesicle transport pathway mediates both autophagosome and melanosome formation, which may involve different regulatory protein complexes. Current studies have revealed that several autophagy-related regulators of autophagosome formation are involved in melanosome formation and maturation and also regulate melanogenesis, and that melanosomes can be degraded via autophagy in melanocytes. Autophagy is also involved in regulating the living environment of melanocytes. Understanding the effects of autophagy on pigmentation may support our understanding of pigmentation diseases. This article reviews the relationship between autophagy and pigmentation in melanocytes.

Signal evolution and morphological complexity in hummingbirds (Aves: Trochilidae).

Understanding how animal signals are produced is critical for understanding their evolution because complexity and modularity in the underlying morphology can affect evolutionary patterns. Hummingbird feathers show some of the brightest and most iridescent colors in nature. These are produced by optically complex stacks of hollow, platelet-shaped organelles called melanosomes. Neither how these morphologies produce colors nor their evolution has been systematically studied. We first used nanoscale morphological measurements and optical modeling to identify the physical basis of color production in 34 hummingbird species. We found that, in general, the melanosome stacks function as multilayer reflectors, with platelet thickness and air space size explaining variation in hue (color) and saturation (color purity). Additionally, light rays reflected from the outer keratin surface interact with those reflected by small, superficial melanosomes to cause secondary reflectance peaks, primarily in short (blue) wavelengths. We then compared variation of both the morphological components and the colors they produce. The outer keratin cortex evolves independently and is more variable than other morphological traits, possibly due to functional constraints on melanosome packing. Intriguingly, shorter wavelength colors evolve faster than longer wavelength colors, perhaps due to developmental processes that enables greater lability of the shapes of small melanosomes. Together, these data indicate that increased structural complexity of feather tissues is associated with greater variation in morphology and iridescent coloration.

TLR3 stimulation induces melanosome endo/phagocytosis through RHOA and CDC42 in human epidermal keratinocyte.

BACKGROUND: Keratinocytes and melanocytes in human epidermis express Toll-like receptors (TLR) and induce immune responses. We previously reported that TLR3 stimulation increases melanosome transport from perinuclear to cell membrane in melanocytes and enhanced release of melanosome from melanocytes, which were followed by increase in melanosome uptake into keratinocytes. OBJECTIVE: In this study, we investigated whether TLR3 stimuli directly affect keratinocytes to enhance melanosome uptake. METHODS: To observe keratinocyte's melanosome uptake ability precisely without melanocytes influences, we isolated melanosomes from human melanocytes and applied isolated melanosomes to keratinocytes stimulated by Poly(I:C). RESULTS: Poly(I:C)-stimulated keratinocytes enhanced uptake of isolated melanosome-rich globules five-times as much as control. Poly(I:C) increases the RNA and protein expressions of RHOA and CDC42, which are small GTP-binding proteins inducing the endocytosis. Pull-down assay showed that Poly(I:C) increased the GTP-binding RHOA and CDC42, suggesting TLR3 stimulation activated RHOA and CDC42. The knockdown of TLR3 suppressed RHOA and CDC42 induction by Poly(I:C). Consistently, the knockdown of RHOA and CDC42 significantly suppressed the melanosome-rich globules uptake by Poly(I:C)-stimulated keratinocytes. CONCLUSION: Because RHOA and CDC42 activation induces endocytosis by modification of actin stress fiber and filopodia formation, respectively, these results suggested that TLR3 stimulation enhances melanosome uptake into keratinocytes through endocytosis mechanisms. Combining with the data of our previous publications, TLR3, which signal is activated by sensing viral molecules, enhance pigmentation by controlling both melanin transport system by RAB GTPases induction in melanocytes and uptake system by RHOA and CDC42 in keratinocytes.

Melanosome maturation proteins Oca2, Mitfa and Vps11 are differentially required for cisplatin resistance in zebrafish melanocytes.

Melanoma is the deadliest form of skin cancer, partially due to its inherent resistance to therapy. Here, we test in live larvae the hypothesis that mature melanosomes contribute to resistance to chemotherapeutic drug, cisplatin, via drug sequestration. We also compare three melanosome biogenesis proteins-microphthalmia-associated transcription factor (Mitfa), vacuolar protein sorting 11 (Vps11) and oculocutaneous albinism 2 (Oca2) to determine their respective contributions to chemoresistance. Melanocytes in zebrafish larvae harbouring loss-of-function mutations in the mitfa, vps11 or oca2 genes are more sensitive to cisplatin damage than wild-type larvae. As a comparison, we examined sensory hair cells of the lateral line, which are sensitive to cisplatin. Hair cells in oca2 and mitfa mutants do not show increased cisplatin sensitivity when compared to wild-type larvae, suggesting the increase in cisplatin sensitivity could be melanocyte specific. However, hair cells in vps11 mutants are more sensitive to cisplatin than their wild-type counterparts, suggesting that this mutation increases cisplatin susceptibility in multiple cell types. This is the first in vivo study to show an increase in chemotherapeutic drug sensitivity when melanosome maturation mutations are present. The proteins tested, especially Oca2, represent novel drug targets for increasing the efficiency of melanoma chemotherapy treatment.

How hormones may modulate human skin pigmentation in melasma: An in vitro perspective.

Melasma is a common acquired hyperpigmentary disorder occurring primarily in photo-exposed areas and mainly affecting women of childbearing age. To decipher the role of sex hormones in melasma, this viewpoint reviews the effects of sex hormones on cutaneous cells cultured in monolayers, in coculture, in 3D models and explants in the presence or the absence of UV. The data show that sex steroid hormones, especially oestrogen, can modulate in vitro pigmentation by stimulating melanocytes and keratinocyte pro-pigmentary factors, but not via fibroblast or mast cell activation. In vitro data suggest that oestrogen acts on endothelial cell count, which may in turn increase endothelin-1 concentrations. However, data on explants revealed that sex steroid even at doses observed during pregnancy cannot induce melanogenesis alone nor melanosome transfer but that it acts in synergy with UVB. In conclusion, we hypothesize that in predisposed persons, sex steroid hormones initiate hyperpigmentation in melasma by amplifying the effects of UV on melanogenesis via direct effects on melanocytes or indirect effects via keratinocytes and on the transfer of melanosomes. They also help to sustain hyperpigmentation by increasing the number of blood vessels and, in turn, the level of endothelin-1.

The absence of Rab27a accelerates the degradation of Melanophilin.

Melanophilin (Mlph) forms an interaction with Rab27a and the actin-based motor protein MyosinVa (MyoVa) on mature melanosome membranes and the tripartite complex regulates melanosome transport in melanocytes. In this study, we found that Rab27a siRNA decreased Mlph and Rab27a protein levels, but Mlph mRNA levels were not changed. Other Rab27a siRNA sequences also showed the same results. When Rab27a siRNA was treated with melan-a melanocytes, Rab27a protein was decreased within 6 hours and Mlph protein was decreased within 24 hours. To determine whether the absence of Rab27a promotes Mlph degradation, we inhibited protein degradation by treatment with proteasome (MG132) and lysosomal enzyme (E64D and Pepstatin A) inhibitors in melan-a melanocytes. MG132 inhibited the degradation of Mlph, but E64D and Pepstatin A had no effect on Mlph. The absence of Rab27a enhanced ubiquitination of Mlph and induced proteasomal degradation. From these results, we concluded that Mlph interaction with Rab27a is important for Mlph stability and melanosome transport.

Metabolic physiology explains macroevolutionary trends in the melanic colour system across amniotes.

Metabolism links organisms to their environment through its effects on thermoregulation, feeding behaviour and energetics. Genes involved in metabolic processes have known pleiotropic effects on some melanic colour traits. Understanding links between physiology and melanic colour is critical for understanding the role of, and potential constraints on, colour production. Despite considerable variation in metabolic rates and presumed ancestral melanic coloration in vertebrates, few studies have looked at a potential relationship between these two systems in a comparative framework. Here, we test the hypothesis that changes in melanosome shape in integumentary structures track metabolic rate variation across amniotes. Using multivariate comparative analyses and incorporating both extant and fossil taxa, we find significantly faster rates of melanosome shape evolution in taxa with high metabolic rates, as well as both colour- and clade-specific differences in the relationship between metabolic rate and melanosome shape. Phylogenetic tests recover an expansion in melanosome morphospace in maniraptoran dinosaurs, as well as rate shifts within birds (in songbirds) and mammals. These findings indicate another core phenotype influenced by metabolic changes in vertebrates. They also provide a framework for testing clade-specific gene expression patterns in the melanocortin system and may improve colour reconstructions in extinct taxa.

no privacy, a Xenopus tropicalis mutant, is a model of human Hermansky-Pudlak Syndrome and allows visualization of internal organogenesis during tadpole development.

We describe a novel recessive and nonlethal pigmentation mutant in Xenopus tropicalis. The mutant phenotype can be initially observed in tadpoles after stage 39/40, when mutant embryos display markedly reduced pigmentation in the retina and the trunk. By tadpole stage 50 almost all pigmented melanophores have disappeared. Most interestingly, those embryos fail entirely to make pigmented iridophores. The combined reduction/absence of both pigmented iridophores and melanophores renders these embryos virtually transparent, permitting one to easily observe both the developing internal organs and nervous system; accordingly, we named this mutant no privacy (nop). We identified the causative genetic lesion as occurring in the Xenopus homolog of the human Hermansky-Pudlak Syndrome 6 (HPS6) gene, combining several approaches that utilized conventional gene mapping and classical and modern genetic tools available in Xenopus (gynogenesis, BAC transgenesis and TALEN-mediated mutagenesis). The nop allele contains a 10-base deletion that results in truncation of the Hps6 protein. In humans, HPS6 is one of the genes responsible for the congenital disease HPS, pathological symptoms of which include oculocutaneous albinism caused by defects in lysosome-related organelles required for pigment formation. Markers for melanin-producing neural crest cells show that the cells that would give rise to melanocytes are present in nop, though unpigmented. Abnormalities develop at tadpole stages in the pigmented retina when overall pigmentation becomes reduced and large multi-melanosomes are first formed. Ear development is also affected in nop embryos when both zygotic and maternal hsp6 is mutated: otoliths are often reduced or abnormal in morphology, as seen in some mouse HPS mutations, but to our knowledge not described in the BLOC-2 subset of HPS mutations nor described in non-mammalian systems previously. The transparency of the nop line suggests that these animals will aid studies of early organogenesis during tadpole stages. In addition, because of advantages of the Xenopus system for assessing gene expression, cell biological mechanisms, and the ontogeny of melanosome and otolith formation, this should be a highly useful model for studying the molecular mechanisms underlying the acquisition of the HPS phenotype and the underlying biology of lysosome-related organelle function.

A melanosomal two-pore sodium channel regulates pigmentation.

Intracellular organelles mediate complex cellular functions that often require ion transport across their membranes. Melanosomes are organelles responsible for the synthesis of the major mammalian pigment melanin. Defects in melanin synthesis result in pigmentation defects, visual deficits, and increased susceptibility to skin and eye cancers. Although genes encoding putative melanosomal ion transporters have been identified as key regulators of melanin synthesis, melanosome ion transport and its contribution to pigmentation remain poorly understood. Here we identify two-pore channel 2 (TPC2) as the first reported melanosomal cation conductance by directly patch-clamping skin and eye melanosomes. TPC2 has been implicated in human pigmentation and melanoma, but the molecular mechanism mediating this function was entirely unknown. We demonstrate that the vesicular signaling lipid phosphatidylinositol bisphosphate PI(3,5)P2 modulates TPC2 activity to control melanosomal membrane potential, pH, and regulate pigmentation.

The consistent difference in red fluorescence in fishes across a 15 m depth gradient is triggered by ambient brightness, not by ambient spectrum.

BACKGROUND: Organisms adapt to fluctuations or gradients in their environment by means of genetic change or phenotypic plasticity. Consistent adaptation across small spatial scales measured in meters, however, has rarely been reported. We recently found significant variation in fluorescence brightness in six benthic marine fish species across a 15 m depth gradient. Here, we investigate whether this can be explained by phenotypic plasticity alone, using the triplefin Tripterygion delaisi as a model species. In two separate experiments, we measure change in red fluorescent brightness to spectral composition and ambient brightness, two central parameters of the visual environment that change rapidly with depth. RESULTS: Changing the ambient spectra simulating light at -5 or -20 m depth generated no detectable changes in mean fluorescence brightness after 4-6 weeks. In contrast, a reduction in ambient brightness generated a significant and reversible increase in mean fluorescence, most of this within the first week. Although individuals can quickly up- and down-regulate their fluorescence around this mean value using melanosome aggregation and dispersal, we demonstrate that this range around the mean remained unaffected by either treatment. CONCLUSION: We show that the positive association between fluorescence and depth observed in the field can be fully explained by ambient light brightness, with no detectable additional effect of spectral composition. We propose that this change is achieved by adjusting the ratio of melanophores and fluorescent iridophores in the iris.

Quantitative characterization and comparative study of feather melanosome internal morphology using surface analysis.

A successful feather development implies in a precise orchestration of cells in the follicle, which culminates in one of the most complex epidermal structures in nature. Melanocytes contribute to the final structure by delivering melanosomes to the barb and barbule cells. Disturbance to the tissue during the feather growth can damage the final structure. Here, melanosomes seen in an unusual outgrowth on the barb cortex of a flight feather are reported and compared to commonly observed melanosomes embedded in the cortex. Transmission Electron Microscopy in scanning-transmission mode (STEM) generated images coupled with secondary electron detection. The two classes of melanosomes were registered on images combining transmitted and secondary electron signals. Image processing allowed surface analyses of roughness and texture of the internal morphology of these organelles. Results showed that the two classes of melanosomes are significantly distinct internally, indicating that different physiological processes up to feather maturation could have occurred. Surface analysis methods are not regularly used in cell biology studies, but here it is shown that it has great potential for microscopic image analysis, which could add robust information to studies of cell biology events.

Trends in nanosecond melanosome microcavitation up to 1540 nm.

Thresholds for microcavitation of bovine and porcine melanosomes were previously reported, using single nanosecond (ns) laser pulses in the visible (532 nm) and the near-infrared (NIR) from 1000 to 1319 nm. Here, we report average radiant exposure thresholds for bovine melanosome microcavitation at additional NIR wavelengths up to 1540 nm, which range from ∼0.159 J∕cm2 at 800 nm to 4.5 J∕cm2 at 1540 nm. Melanosome absorption coefficients were also estimated, and decreased with increasing wavelength. These values were compared to retinal pigment epithelium coefficients, and to water absorption, over the same wavelength range. Corneal total intraocular energy retinal damage threshold values were estimated and compared to the previous (2007) and recently changed (2014) maximum permissible exposure (MPE) safe levels. Results provide additional data that support the recent changes to the MPE levels, as well as the first microcavitation data at 1540 nm, a wavelength for which melanosome microcavitation may be an ns-pulse skin damage mechanism.

Interpreting melanin-based coloration through deep time: a critical review.

Colour, derived primarily from melanin and/or carotenoid pigments, is integral to many aspects of behaviour in living vertebrates, including social signalling, sexual display and crypsis. Thus, identifying biochromes in extinct animals can shed light on the acquisition and evolution of these biological traits. Both eumelanin and melanin-containing cellular organelles (melanosomes) are preserved in fossils, but recognizing traces of ancient melanin-based coloration is fraught with interpretative ambiguity, especially when observations are based on morphological evidence alone. Assigning microbodies (or, more often reported, their 'mouldic impressions') as melanosome traces without adequately excluding a bacterial origin is also problematic because microbes are pervasive and intimately involved in organismal degradation. Additionally, some forms synthesize melanin. In this review, we survey both vertebrate and microbial melanization, and explore the conflicts influencing assessment of microbodies preserved in association with ancient animal soft tissues. We discuss the types of data used to interpret fossil melanosomes and evaluate whether these are sufficient for definitive diagnosis. Finally, we outline an integrated morphological and geochemical approach for detecting endogenous pigment remains and associated microstructures in multimillion-year-old fossils.

A guide to the field of palaeo colour: Melanin and other pigments can fossilise: Reconstructing colour patterns from ancient organisms can give new insights to ecology and behaviour.

Melanin, and other pigments have recently been shown to preserve over geologic time scales, and are found in several different organisms. This opens up the possibility of inferring colours and colour patterns ranging from invertebrates to feathered dinosaurs and mammals. An emerging discipline is palaeo colour: colour plays an important role in display and camouflage as well as in integumental strengthening and protection, which makes possible the hitherto difficult task of doing inferences about past ecologies, behaviours, and organismal appearance. Several studies and techniques have been presented in the last couple of years that have described ways to characterize pigment patterns. Here, I will review the available methods and the likely applications to understand past ecologies. A golden age of colourized dinosaurs and other animals is now dawning upon us, which may elucidate the nature of ancient predator prey interactions and display structures.

Orientation of actin filaments in teleost retinal pigment epithelial cells, and the effect of the lectin, Concanavalin A, on melanosome motility.

Retinal pigment epithelial cells of teleosts contain numerous melanosomes (pigment granules) that exhibit light-dependent motility. In light, melanosomes disperse out of the retinal pigment epithelium (RPE) cell body (CB) into long apical projections that interdigitate with rod photoreceptors, thus shielding the photoreceptors from bleaching. In darkness, melanosomes aggregate through the apical projections back into the CB. Previous research has demonstrated that melanosome motility in the RPE CB requires microtubules, but in the RPE apical projections, actin filaments are necessary and sufficient for motility. We used myosin S1 labeling and platinum replica shadowing of dissociated RPE cells to determine actin filament polarity in apical projections. Actin filament bundles within RPE apical projections are uniformly oriented with barbed ends toward the distal tips. Treatment of RPE cells with the tetravalent lectin, Concanavalin A, which has been shown to suppress cortical actin flow by crosslinking of cell-surface proteins, inhibited melanosome aggregation and stimulated ectopic filopodia formation but did not block melanosome dispersion. The polarity orientation of F-actin in apical projections suggests that a barbed-end directed myosin motor could effect dispersion of melanosomes from the CB into apical projections. Inhibition of aggregation, but not dispersion, by ConA confirms that different actin-dependent mechanisms control these two processes and suggests that melanosome aggregation is sensitive to treatments previously shown to disrupt actin cortical flow.

Melanocytes and their diseases.

Human melanocytes are distributed not only in the epidermis and in hair follicles but also in mucosa, cochlea (ear), iris (eye), and mesencephalon (brain) among other tissues. Melanocytes, which are derived from the neural crest, are unique in that they produce eu-/pheo-melanin pigments in unique membrane-bound organelles termed melanosomes, which can be divided into four stages depending on their degree of maturation. Pigmentation production is determined by three distinct elements: enzymes involved in melanin synthesis, proteins required for melanosome structure, and proteins required for their trafficking and distribution. Many genes are involved in regulating pigmentation at various levels, and mutations in many of them cause pigmentary disorders, which can be classified into three types: hyperpigmentation (including melasma), hypopigmentation (including oculocutaneous albinism [OCA]), and mixed hyper-/hypopigmentation (including dyschromatosis symmetrica hereditaria). We briefly review vitiligo as a representative of an acquired hypopigmentation disorder.